Notch Signaling Is Activated in Human Hepatocellular Carcinoma and Induces Tumor Formation in Mice

doi:10.1053/j.gastro.2012.09.002

Gastroenterology

Volume 143, Issue 6, December 2012, Pages 1660-1669.e7

https://doi.org/10.1053/j.gastro.2012.09.002 Get rights and content

Background & Aims

The Notch signaling pathway is activated in leukemia and solid tumors (such as lung cancer), but little is known about its role in liver cancer.

Methods

The intracellular domain of Notch was conditionally expressed in hepatoblasts and their progeny (hepatocytes and cholangiocytes) in mice. This was achieved through Cre expression under the control of an albumin and α-fetoprotein (AFP) enhancer and promoter (AFP-Notch intracellular domain [NICD]). We used comparative functional genomics to integrate transcriptome data from AFP-NICD mice and human hepatocellular carcinoma (HCC) samples (n = 683). A Notch gene signature was generated using the nearest template prediction method.

Results

AFP-NICD mice developed HCC with 100% penetrance when they were 12 months old. Activation of Notch signaling correlated with activation of 3 promoters of insulin-like growth factor 2; these processes appeared to contribute to hepatocarcinogenesis. Comparative functional genomic analysis identified a signature of Notch activation in 30% of HCC samples from patients. These samples had altered expression in Notch pathway genes and activation of insulin-like growth factor signaling, despite a low frequency of mutations in regions of NOTCH1 associated with cancer. Blocking Notch signaling in liver cancer cells with the Notch activation signature using γ-secretase inhibitors or by expressing a dominant negative form of mastermind-like 1 reduced their proliferation in vitro.

Conclusions

Notch signaling is activated in human HCC samples and promotes formation of liver tumors in mice. The Notch signature is a biomarker of response to Notch inhibition in vitro.

Section snippets

Materials and Methods

For additional information, please see Supplementary Materials and Methods.

Transcript profiling: Submitted microarray data accession numbers are GSE33486 and GSE33560 (GEO Omnibus: http://www.ncbi.nlm.nih.gov/geo/).

Liver-Specific Activation of Notch Pathway Promotes Oncogenesis

We sought to evaluate long-term effects of Notch signaling in vivo by using bigenic AFP-NICD mice, in which Cre-mediated recombination in embryonic hepatoblasts results in the expression of a constitutively active form of Notch1 in > 95% of hepatoblasts and cholangiocytes postnatally⁶ (Figure 1A). At 6 months of age, AFP-NICD mice developed liver tumors at an estimated frequency of 33% (1/3) and 50% (2/4) at 9 months. Beyond 12 months, all AFP-NICD mice showed macroscopic liver tumors (12/12,

Discussion

In this study, we have undertaken a comprehensive and integrative approach to explore the role of Notch signaling in liver cancer pathogenesis. We have found that Notch signaling promotes liver carcinogenesis in a genetically engineered mouse model and that this pathway is activated in one third of human HCCs. Specifically, our studies demonstrate that (1) liver-specific Notch activation in mice recapitulates features of human hepatocarcinogenesis, including dysplasia and HCC; (2) several genes

Acknowledgments

The authors thank Warren Pear and Kristin Toscano for the generous gift of DN-MAML and GFP control virus and Archana Panikkar for help with initial studies of AFP-NICD tumors.

A.V., C.A., and K.Y. contributed equally to this work.

References (48)

J.M. Llovet et al.
Hepatocellular carcinoma
Lancet
(2003)
C. Wang et al.
Notch1 signaling sensitizes tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human hepatocellular carcinoma cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression
J Biol Chem
(2009)
C. Giovannini et al.
Selective ablation of Notch3 in HCC enhances doxorubicin's death promoting effect by a p53 dependent mechanism
J Hepatol
(2009)
A. Villanueva et al.
Combining clinical, pathology, and gene expression data to predict recurrence of hepatocellular carcinoma
Gastroenterology
(2011)
I. Maillard et al.
Canonical notch signaling is dispensable for the maintenance of adult hematopoietic stem cells
Cell Stem Cell
(2008)
V. Tovar et al.
IGF activation in a molecular subclass of hepatocellular carcinoma and pre-clinical efficacy of IGF-1R blockage
J Hepatol
(2010)
G. Nardone et al.
Activation of fetal promoters of insulin-like growth factors II gene in hepatitis C virus-related chronic hepatitis, cirrhosis, and hepatocellular carcinoma
Hepatology
(1996)
W. Guo et al.
Slug and Sox9 cooperatively determine the mammary stem cell state
Cell
(2012)
S. Toffanin et al.
MicroRNA-based classification of hepatocellular carcinoma and oncogenic role of miR-517a
Gastroenterology
(2011)
M. Liu et al.
IKKα; activation of NOTCH links tumorigenesis via FOXA2 Suppression
Mol Cell
(2012)

A. Jemal et al.

Global cancer statistics

CA Cancer J Clin

(2011)

S.F. Altekruse et al.

Hepatocellular carcinoma incidence, mortality, and survival trends in the United States from 1975 to 2005

J Clin Oncol

(2009)

J.M. Llovet et al.

Sorafenib in advanced hepatocellular carcinoma

N Engl J Med

(2008)

K.S. Zaret

Genetic programming of liver and pancreas progenitors: lessons for stem-cell differentiation

Nat Rev Genet

(2008)

Y. Zong et al.

Notch signaling controls liver development by regulating biliary differentiation

Development

(2009)

P. Ranganathan et al.

Notch signalling in solid tumours: a little bit of everything but not all the time

Nat Rev Cancer

(2011)

B. Westhoff et al.

Alterations of the Notch pathway in lung cancer

Proc Natl Acad Sci U S A

(2009)

S. Santagata et al.

JAGGED1 expression is associated with prostate cancer metastasis and recurrence

Cancer Res

(2004)

R. Qi et al.

Notch1 signaling inhibits growth of human hepatocellular carcinoma through induction of cell cycle arrest and apoptosis

Cancer Res

(2003)

L.C. Murtaugh et al.

Notch signaling controls multiple steps of pancreatic differentiation

Proc Natl Acad Sci U S A

(2003)

C. Kellendonk et al.

Hepatocyte-specific expression of Cre recombinase

Genesis

(2000)

D.Y. Chiang et al.

Focal gains of VEGFA and molecular classification of hepatocellular carcinoma

Cancer Res

(2008)

E. Wurmbach et al.

Genome-wide molecular profiles of HCV-induced dysplasia and hepatocellular carcinoma

Hepatology

(2007)

Y. Hoshida et al.

Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma

Cancer Res

(2009)

Cited by (250)

Emerging trends in gastrointestinal cancers: Targeting developmental pathways in carcinogenesis and tumor progression
2024, International Review of Cell and Molecular Biology
Gastrointestinal carcinomas are a group of cancers associated with the digestive system and its accessory organs. The most prevalent cancers related to the gastrointestinal tract are colorectal, gall bladder, gastric, hepatocellular, and esophageal cancers, respectively. Molecular aberrations in different signaling pathways, such as signal transduction systems or developmental pathways are the chief triggering mechanisms in different cancers Though a massive advancement in diagnostic and therapeutic interventions results in improved survival of patients with gastrointestinal cancer; the lower malignancy stages of these carcinomas are comparatively asymptomatic. Various gastrointestinal-related cancers are detected at advanced stages, leading to deplorable prognoses and increased rates of recurrence. Recent molecular studies have elucidated the imperative roles of several signaling pathways, namely Wnt, Hedgehog, and Notch signaling pathways, play in the progression, therapeutic responsiveness, and metastasis of gastrointestinal-related cancers. This book chapter gives an interesting update on recent findings on the involvement of developmental signaling pathways their mechanistic insight in gastrointestinalcancer. Subsequently, evidences supporting the exploration of gastrointestinal cancer related molecular mechanisms have also been discussed for developing novel therapeutic strategies against these debilitating carcinomas.
Sublethal necroptosis signaling promotes inflammation and liver cancer
2023, Immunity
It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged “sublethal” state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma.
Treatment of HCC with claudin-1-specific antibodies suppresses carcinogenic signaling and reprograms the tumor microenvironment
2023, Journal of Hepatology
Citation Excerpt :
Since we did not observe a significant induction of Notch effector genes in the PDX HCC transcriptome LI6716 (Fig. 5A), it is conceivable that Notch signaling is either an early event in the signal cascade or that perturbation of Notch signaling is heterogenous in individual tumors. The Notch pathway has been identified as a key regulator of cell differentiation, fate and survival and several functional and clinical studies have shown that the Notch pathway plays a role in the pathogenesis of HCC.16,17 Since Notch signaling has also been identified as a regulator of innate and adaptive immune responses,18 its perturbation by CLDN1 mAb treatment may also contribute to the observed effects on the TIME (Fig. 7).
Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored.
Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC.
Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment.
Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC.
Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.
Is liver regeneration key in hepatocellular carcinoma development?
2023, Trends in Cancer
The liver is the largest organ of the mammalian body and has the remarkable ability to fully regenerate in order to maintain tissue homeostasis. The adult liver consists of hexagonal lobules, each with a central vein surrounded by six portal triads localized in the lobule border containing distinct parenchymal and nonparenchymal cells. Because the liver is continuously exposed to diverse stress signals, several sophisticated regenerative processes exist to restore its functional status following impairment. However, these stress signals can affect the liver’s capacity to regenerate and may lead to the development of hepatocellular carcinoma (HCC), one of the most aggressive liver cancers. Here, we review the mechanisms of hepatic regeneration and their potential to influence HCC development.
Research progress on the role of cholesterol in hepatocellular carcinoma
2023, European Journal of Pharmacology
Hepatocellular carcinoma (HCC) is often diagnosed at advanced stages with no effective treatment options. Mechanistically, it is a complex biological process. Recently, the main cause of its incidence is changing from viral to non-viral. It has been shown that high cholesterol levels can cause the further transformation of non-alcoholic fatty liver disease (NAFLD) to HCC, but some investigations have found that serum cholesterol levels are negatively correlated with morbidity and mortality. Conflicting experimental results and epidemiological investigations illustrate the complex mechanisms of HCC. Cholesterol is essential for the survival of the body and tumors, although research on the function of cholesterol in tumors is evolving, the use of lowering cholesterol drugs in treating HCC remains limited. In this review, the cholesterol-involved mechanisms that cause the development of HCC or reduce the mortality and the latest progress in the use of cholesterol in the treatment of HCC and prospects for prevention and diagnosis have been summarized.
An overview of hepatocellular carcinoma stem cells and potential targeted therapies
2023, Cancer Stem Cells and Signaling Pathways
Hepatocellular carcinoma (HCC) is thought to be the leading cause of cancer-related death in the world. The paucity of blood biomarkers for diagnosis, prognosis, and treatment response prediction further complicates the development of customized therapeutics. Although the most advanced HCC therapeutic approaches can target mature cancer tissues with radiotherapy, chemotherapy, targeted drugs, and other traditional techniques, they are unable to target cancer stem cells (CSCs). CSCs are subpopulation of cells with the ability to self-renew differentiation, and plasticity. They are able to evade treatment, self-renew to form CSC pools, resuscitate cancer further, and metastasize to various tissue sites. To date, liver CSCs have emerged as a promising therapeutic target for HCC. There are several surface markers for liver CSCs, including CD133, CD90, CD44, CD13, and epithelial cell adhesion molecules. These surface markers function as therapeutic targets for eradicating these cells. Multiple therapeutic strategies are being developed to target CSCs. CSCs can be effectively attacked by targeting many signaling cascades such as Janus kinase/signal transducers and activators of transcription, Akt (protein kinase B, PKB), Hedgehog, Notch, transforming growth factor beta, Wnt, and nuclear factor kappa-light-chain-enhancer of activated B cells pathways. Therefore CSCs can be inhibited by targeting these self-renewal mechanisms, which limit stem cell division and differentiation. Significant progress has recently occurred in the investigation of inhibitors of CSC self-renewal mechanisms in HCC. As a result, the main focus of this chapter is summarizing the classification of the CSCs, investigating their potential role in HCC-targeted therapy, and emphasizing their importance in the HCC therapy.

View all citing articles on Scopus

: Conflicts of interest The authors disclose no conflicts.

: Funding C.A. is supported by a grant from Instituto de Salud Carlos III (ISCIII/FIS FI09/00605). J.M.L. is supported by grants from the US National Institute of Diabetes and Digestive and Kidney Diseases (J.M.L: 1R01DK076986), European Commission Framework Programme 7 (HEPTROMIC, Proposal No: 259744), The Samuel Waxman Cancer Research Foundation, the Spanish National Health Institute (J.M.L: SAF-2010-16055), and the Asociación Española Contra el Cáncer. The study was supported by the Landon Foundation - American Association for Cancer Research Innovator Award for International Collaboration in Cancer Research. B.Z.S. is supported by US National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK083355 and DP2-DK083111) and a grant from the Pew Charitable Trusts.

View full text

Original ResearchBasic and Translational—LiverNotch Signaling Is Activated in Human Hepatocellular Carcinoma and Induces Tumor Formation in Mice

Background & Aims

Methods

Results

Conclusions

Section snippets

Materials and Methods

Liver-Specific Activation of Notch Pathway Promotes Oncogenesis

Discussion

Acknowledgments

Lancet

J Biol Chem

J Hepatol

Gastroenterology

Cell Stem Cell

J Hepatol

Hepatology

Cell

Gastroenterology

Mol Cell

Global cancer statistics

CA Cancer J Clin

Hepatocellular carcinoma incidence, mortality, and survival trends in the United States from 1975 to 2005

J Clin Oncol

Sorafenib in advanced hepatocellular carcinoma

N Engl J Med

Genetic programming of liver and pancreas progenitors: lessons for stem-cell differentiation

Nat Rev Genet

Notch signaling controls liver development by regulating biliary differentiation

Development

Notch signalling in solid tumours: a little bit of everything but not all the time

Nat Rev Cancer

Alterations of the Notch pathway in lung cancer

Proc Natl Acad Sci U S A

JAGGED1 expression is associated with prostate cancer metastasis and recurrence

Cancer Res

Notch1 signaling inhibits growth of human hepatocellular carcinoma through induction of cell cycle arrest and apoptosis

Cancer Res

Notch signaling controls multiple steps of pancreatic differentiation

Proc Natl Acad Sci U S A

Hepatocyte-specific expression of Cre recombinase

Genesis

Focal gains of VEGFA and molecular classification of hepatocellular carcinoma

Cancer Res

Genome-wide molecular profiles of HCV-induced dysplasia and hepatocellular carcinoma

Hepatology

Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma

Cancer Res

Original Research
Basic and Translational—Liver
Notch Signaling Is Activated in Human Hepatocellular Carcinoma and Induces Tumor Formation in Mice